va apropar les mans

entenia la situació dels joves

arribada la circumstància

entenia les coses

feia goig, era bonica

part de la casa on dinem, mirem la tele, llegim

astut, hàbil

aixecant

fer teatre, no actuar de forma sincera

va!

estratègia màgica

pensat, planificat

flirtegen, estan interessats a tenir una relació

pesats, insistents

vostè és

em va dir

algú que no forma part de la comunitat, de fora

no prestar atenció.

demano si us plau

amabilitat, bona disposició

seriós

un aire, una certa actitud

l'entrada de la casa

de la paret

mirant pel costat de l'ull

s'equivocava

necessita

Disculpi

bata d'anar per casa

patilles

em cuidava

confiava

tocar temptativament amb les mans

ràpidament

contagiava

entengui

és millor

portar (una peça de roba, un "look") per primera vegada

totalment

que fa quelcom amb pressa, ràpidament

llaminadures

animal

captivitat, no llibertat

més fàcil de passar, més tolerable

dèbil

va ser

entusiasme, il·lusió

ni fred ni calent, tebi

tombarelles, girs

em feia bella, bonica

quan fan la seva feina

intentant no fer-ho de forma òbvia

es veia, tenia aparença de

excusant-me

feia bon aspecte, estava guapo

m'agrada, em satisfà

va ser

algú

Exactly, it’s not about what’s “right” or “wrong.” It’s about understanding when to be casual and when to be formal. That’s a skill, not a rule.

Funny example but it makes sense. It shows how easily you can switch tones once you know your audience.

This lesson is smart, it helps students realize they already switch how they talk depending on the situation. She’s just giving that a name.

This is her main point: students can use both styles, but they need to understand which one fits where. That’s what real communication skills are about.

She’s explaining why texting shows up in schoolwork, it’s what students do all day. It’s not bad writing, just habit, and that’s an important difference.

She realizes her students don’t see texting as wrong. It’s just how people communicate now, which changes how teachers have to approach writing.

Regularization is a technique that penalizes covariates when we have a lot of features. It either shrinks covariates or zeros out covariates completely. For smoother function we want a larger penalty term to shrink coefficients/

I think this line really captures why Silicon Valley is so confusing. There's this mix of idealism and business that doesn't quite fit together. As someone who's grown up here and become more interested in business, I've seen this firsthand. People talk about changing the world and making things better for everyone, but at the same tmie they're constantly chasing investors and profits. They want to seem different from traditional corporations, but in the end, they still just want money. It's visible everywhere today. AI companies claim to be ethical while competing to dominate the market. Startups talk about helping people but really just want funding and profit. Marwick's point about Web 2.0 being born out of activism and capitalism explains that contradiction so well. It's what makes Silicon Valley interesting but also kind of fake. It's built on a constant clash between wanting to do good and wanting to make money.

I found it interesting how this line really captures the optimism that surrounded social media in its early days. Everyone genuinely believed it could change the world, especially the way that we communicate. It's wild to think about how sincere that excitement was compared to now. Even though platforms like TikTok and Instagram still connect to millions of people and let them share their lives, the focus has shifted. What used to feel like a happy place for community has turned into something more performative and sometimes even toxic. People's attitudes toward social media have gone from excitement about connection to serious concerns about mental health, misinformation, and negativity. It's often seen now as one of the biggest causes of anxiety, depression, and even suicide, especially among teens. I think the way that Marwick includes this quote really sets the tone for her critique. It reminds readers of what social media was supposed to be before showing what it's actually become.

Které části textu výše vysvětlují, ke kterým morálním soustavám vede kapitalismus? Měl autor na mysli tuto pasáž?

Kapitalizmus stavia samého seba do kontrastu s agresívnymi totalitami, aby skryl vlastnú plíživú totalitárnosť a mohol sa tváriť ako nie dokonalý, no najlepší možný systém. V kombinácii s demokratickými ideálmi sa odieva do plášťa garanta dodržiavania univerzálnych ľudských práv, ktoré sú pre neho však iba nadstavbou. Je kompatibilný s akoukoľvek sústavou morálnych hodnôt a preto sa nevylučuje ani s fašizmom. Práve naopak. Mechanizmy, ktoré zavádza, rozsievajú semienka fašistických tendencií. Tie klíčia na ním zoranej pôde a napokon vyrastú do podoby, v ktorej sa začnú kriticky veľkej časti spoločnosti javiť ako prijateľná alternatíva k nedôstojným pomerom, do ktorých ju paradoxne uvrhla sama kapitalistická mašinéria.

Lidé odevzdávají moc diktátorům a korporátním mafiím, když volí zkorumpované politiky.

"household-"

This is minor but should this be step #5? Then the list of 9.1-9.5 nicely aligns with the numbered steps here

Consider reformatting this into a bulleted list

Remove italics to be consistent with other sub headers

Change to "are" for consistent present tense

"Residents of group quarters," since the examples cite group quarters, not residents

Should this have additional guidance?

I suggest rephrasing, something like: "data so that estimates align with the targets (i.e.,population)."

typo

I love showing the students how this works with the students when they have different answers to problems or differences in opinions. We discuss the different ideas and dissect or unpack the ideas together and see if one answer has a better reason than the other. This helps the students understand how everyone thinks differently and comes to their own conclusions differently. Some of the responses from the students are mind blowing and fantastic rationals due to personal experiences.

bu başlıkta sorun var

deneme

I feel like this section is very important for the article, but there is no citation supporting these claims.

None of the images have citations, we don't know where these pictures came from (for example, a museum colection). Also, are these images folowing the Wikipedia's copyright guidlines?

There is no citation in this section. If you are planning on editing the article, maybe it would be a good idea to find sources to support these claims.

Just like we talk in class, there are many claims with no citation in this section.

This may be splitting hairs. Outside the study region is listed as "gray" here but in the figure & legend, it appears more purple. Maybe because it's the rainbow colors which make me assume the last color is purple (or violet), and also because there are other regions in the map that are gray, albeit a lighter shade of gray

Can this be displayed within the paragraph and not in this separate text box? It's odd to have to scroll horizontally to finish reading this paragraph

Puisque l'idée du web était de pouvoir accéder à des fichiers présents sur d'autres ordinateurs à travers des adresse uniques et en utilisant l'infrastructure d'internet pour y accéder.

This is a concept we need to explain more clearly to everyone—especially minors. Many don’t realize that even if you're underage yourself, sharing or possessing certain types of photos involving others can still be illegal and harmful. It’s not just about personal choices; it’s about understanding consent, privacy, and the law.

This is a concept I wish had been taught earlier. A lot of people post things when they’re young that can reflect poorly on them later. I didn’t learn about this until my senior year, when we were taught that colleges and employers might look at your social media to get a sense of who you are. It really made me think about how important it is to be intentional with what we share online.

I agree this is an important practice to teach our students. Photos and videos taken at school are a big part of their daily lives, but they don’t always consider who might be in the background or whether that person would want to be publicly shared. Helping students think critically about consent and privacy is essential.

for Trump corruption - inauguration bribes

Definition.

Definition.

Look into this project and at a small summary about what it is here...

super interesante un acercamiento a la cultura asiatica vía las humanidades digitales

Ferenc József császár adta ki 1849. március 4-én Olmützben.

autonómia**

This has always been a question of him, how have we developed different cultures, and how does everyone not follow the same one?

It is interesting how they are telling us how winking requires more effort; which is completely true because it does need more effort and sometimes people cannot do it.

This field of anthropology is the most interesting to me because I find it interesting how we can learn how ancient people have lived.

Since most of Americans have house holds that are held together buy animals skins and sandstone. Its kind of interesting how some are still not as advanced as us.

It still amazes me that this was the original standard. So much has changed over time.

I think the argument being made here, is that just having a price by itself it's a gross oversimplification about what is needed.

And just because a price has been useful in places where you have seen a transition away from fossil fuel use, it doesn't mean you should start with a price.

The price is the thing that comes in once you have a clear alternative to the fossil-powered default, to make it less attractive.

I think this is quite helpful as the simplistic version of the argument about why a carbon market by itself is not good enough. You can also make a similar argument for technical carbon removal. it's just too expensive to rely on.

I think the argument here is that, you need to build the alternative before you can choke off the fossil incumbent.

A precondition of being able to raise prices is having something people can migrate TO, that meets their needs. Otherwise, people reject the premise of a lot of the time,

This seem to be arguing that carbon pricing is really ineffective in the face of inelastic demand. You can point the gillet jaune in France and their protests about the price of diesel was a good example of this. The demand inelasticity thing though is extremely important to get your head around, because while there are definitely clear examples that we have seen there are also many four cases where it's much less clear cut and things may indeed be more elastic than we thought.

So a key thing about the global covered market discussion at cop, is that there is no single Price. So just like our countries compete to have low tax resumes you have the same thing for carbon here. Oh dear.

Wow, Uruguay actually has a high carbon tax?

The correct order is: Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit; Marisa Senngam. This will be changed in the next revision and the final manuscript.

The correct order is: Suphannika Pornwattanakavee; Nattawut Leelakanok; Teerarat Todsarot; Gabrielle Angele Tatta Guinto; Ratchanon Takun; Assadawut Sumativit; Marisa Senngam. This will be changed in the next revision and the final manuscript.

Paine wanted everyone equal. His idea of America withough Britain's control over them was something a lot of people may not have been ready for.

Paine addressed "Common Sense" to ordinary colonists his emotional style helped turn revolutionary ideas into later starting the Declaration of Independence.

Looking at this now in our world, do you think Paine had a different idea for freedom?

How does his definition of equality differ from in the Declaration of Independence? Paine's hope was for women and men of all races and ethnicities to be equal. This was not the case.

Paine wrote the pamphlet called, "Common Sense" to persuade other Americans to break away from Britain.

Thomas Paine used religious language to intrigue the colonists. In 1776, religious authority was very respected in the political world. Him saying the monarchy was from the Devil was a smart move for him since he was for independence and everyone valued religion at the time.

Necessary evil is used to describe the government as something important for the people to have so their rights aren't harmed.

A monarchy is a form of government where a single person governs the head of state.

why did Blake emphasize that not all articles are about standard English?

eLife Assessment

This study provides useful insights into the ways in which germinal center B cell metabolism, particularly lipid metabolism, affects cellular responses. The authors use sophisticated mouse models to demonstrate that ether lipids are relevant for B cell homeostasis and efficient humoral responses. Although the data were collected from in vitro and in vivo experiments and analyzed using solid and validated methodology, more careful experiments and extensive revision of the manuscript will be required to strengthen the authors' conclusions.

Reviewer #1 (Public review):

In this manuscript, Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.

Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.

Highlights of the Manuscript:

The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.

The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.

Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.

Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells. However, several major points need to be addressed:

Major Comments:

Figures 1 and 2

The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).

The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.

Suggestions for Improvement:

- B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.

- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.<br /> The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.

- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.

- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.

Reviewer #2 (Public review):

Summary:

In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.

Strengths:

Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.

Weaknesses:

More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.

Author response:

eLife Assessment

This study provides useful insights into the ways in which germinal center B cell metabolism, particularly lipid metabolism, affects cellular responses. The authors use sophisticated mouse models to demonstrate that ether lipids are relevant for B cell homeostasis and efficient humoral responses. Although the data were collected from in vitro and in vivo experiments and analyzed using solid and validated methodology, more careful experiments and extensive revision of the manuscript will be required to strengthen the authors' conclusions.

In addition to praise for the eLife system and transparency (public posting of the reviews; along with an opportunity to address them), we are grateful for the decision of the Editors to select this submission for in-depth peer review and to the referees for the thoughtful and constructive comments.

In overview, we mostly agree with the specific comments and evaluation of strengths of what the work adds as well as with indications of limitations and caveats that apply to the breadth of conclusions. One can view these as a combination of weaknesses, of instances of reading more into the work than what it says, and of important future directions opened up by the findings we report. Regarding the positives, we appreciate the reviewers' appraisal that our work unveils a novel mechanism in which the peroxisomal enzyme PexRAP mediates B cell intrinsic ether lipid synthesis and promotes a humoral immune response. We are gratified by a recognition that a main contribution of the work is to show that a spatial lipidomic analysis can set the stage for discovery of new molecular processes in biology that are supported by using 2-dimensional imaging mass spectrometry techniques and cell type specific conditional knockout mouse models.

By and large, the technical issues are items we will strive to improve. Ultimately, an over-arching issue in research publications in this epoch are the questions "when is enough enough?" and "what, or how much, advance will be broadly important in moving biological and biomedical research forward?" It appears that one limitation troubling the reviews centers on whether the mechanism of increased ROS and multi-modal death - supported most by the in vitro evidence - applies to germinal center B cells in situ, versus either a mechanism for decreased GC that mostly applies to the pre-GC clonal amplification (or recruitment into GC). Overall, we agree that this leap could benefit from additional evidence - but as resources ended we instead leave that question for the future other than the findings with S1pr2-CreERT2-driven deletion leading to less GC B cells. While we strove to be very careful in framing such a connection as an inference in the posted manuscript, we will revisit the matter via rechecking the wording when revising the text after trying to get some specific evidence.  

In the more granular part of this provisional response (below), we will outline our plan prompted by the reviewers but also comment on a few points of disagreement or refinement (longer and more detailed explanation). The plan includes more detailed analysis of B cell compartments, surface level of immunoglobulin, Tfh cell population, a refinement of GC B cell markers, and the ex vivo GC B cell analysis for ROS, proliferation, and cell death. We will also edit the text to provide more detailed information and clarify our interpretation to prevent the confusion of our results.  At a practical level, some evidence likely is technologically impractical, and an unfortunate determinant is the lack of further sponsored funding for further work. The detailed point-by-point response to the reviewer’s comments is below.  

Public Reviews:

Reviewer #1 (Public review):

In this manuscript, Sung Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.

Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.

Highlights of the Manuscript:

The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.

The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.

Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.

Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells.

We appreciate these positive reactions and response, and agree with the overview and summary of the paper's approaches and strengths.

However, several major points need to be addressed:

Major Comments:

Figures 1 and 2

The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).

The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.

Suggestions for Improvement:

B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.

Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we believe we will be able to polish a revised manuscript through addition of results of analyses suggested by this point in the review: measurement of surface IgM on and phenotyping of various B cell subsets, including MZB and B1 B cells, to extend the data in Supplemental Fig 1H and I. Depending on the level of support, new immunization experiments to score Tfh and analyze a few of their functional molecules as part of a B cell paper may be feasible.  

- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.

The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.

We first want to apologize the mislabeling of LZ and DZ in Fig 1H. The greenish-yellow colored region (GL7⁺ CD35⁺) indicate the DZ and the cyan-colored region (GL7⁺ CD35⁺) indicates the LZ.

As a technical note, we experienced high background noise with GL7 staining uniquely with PexRAP deficient (Dhrs7b^f/f; Rosa26-CreER^T2) mice (i.e., not WT control mice). The high background noise of GL7 staining was not observed in B cell specific KO of PexRAP (Dhrs7b^f/f; huCD20-CreER^T2). Two formal possibilities to account for this staining issue would be if either the expression of the GL7 epitope were repressed by PexRAP or the proper positioning of GL7⁺ cells in germinal center region were defective in PexRAP-deficient mice (e.g., due to an effect on positioning cues from cell types other than B cells). In a revised manuscript, we will fix the labeling error and further discuss the GL7 issue, while taking care not to be thought to conclude that there is a positioning problem or derepression of GL7 (an activation antigen on T cells as well as B cells).

While the gating strategy for an overall population of GC B cells is fairly standard even in the current literature, the question about using CD138 staining to exclude early plasmablasts (i.e., analyze B220⁺ CD138^neg vs B220⁺ CD138⁺) is interesting. In addition, some papers like to use GL7⁺ CD38^neg for GC B cells instead of GL7⁺ Fas (CD95)⁺, and we thank the reviewer for suggesting the analysis of centroblasts and centrocytes. For the revision, we will try to secure resources to revisit the immunizations and analyze them for these other facets of GC B cells (including CXCR4/CD86) and for their GL7⁺ CD38^neg. B220⁺ CD138^- and B220⁺ CD138⁺ cell populations. 

We agree that comparison of the Rosa26-CreERT2 results to those with B cell-specific loss-of-function raise a tantalizing possibility that Tfh cells also are influenced by PexRAP. Although the manuscript is focused on post-ontogenic B cell regulation in Ab responses, we hope to add a new immunization experiments that scores Tfh and analyzes a few of their functional molecules could be added to this B cell paper, depending on the ability to wheedle enough support / fiscal resources.

- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.

In the revision, we will edit the text and try to adjust the digitized cytometry data to allow more dynamic range to the right side of the upper panels in Fig. 2E, and otherwise to improve the presentation of the in vivo CTV result. However, we feel impelled to push back respectfully on some of the concern raised here. First, it seems to gloss over the presentation of multiple facets of evidence. The conclusion about maintenance derives primarily from Fig. 2C, which shows a rapid, statistically significant decrease in B cell numbers (extending the finding of Fig. 1D, a more substantial decrease after a bit longer a period). As noted in the text, the rate of de novo B cell production does not suffice to explain the magnitude of the decrease.

In terms of proliferation, we will improve presentation of the Methods but the bottom line is that the recovery efficiency is not bad (comparing to prior published work) inasmuch as transferred B cells do not uniformly home to spleen. In a setting where BAFF is in ample supply in vivo, we transferred equal numbers of cells that were equally labeled with CTV and counted B cells.  The CTV result might be affected by lower recovered B cell with PexRAP deficiency, generally, the frequencies of CTV^low divided population are not changed very much. However, it is precisely because of the pitfalls of in vivo analyses that we included complementary data with survival and proliferation in vitro. The proliferation was attenuated in PexRAP-deficient B cells in vitro; this evidence supports the conclusion that proliferation of PexRAP knockout B cells is reduced. It is likely that PexRAP deficient B cells also have defect in viability in vivo as we observed the reduced B cell number in PexRAP-deficient mice. As the reviewer noticed, the presence of a defect in cycling does, in the transfer experiments, limit the ability to interpret a lower yield of B cell population after adoptive transfer into µMT recipient mice as evidence pertaining to death rates. We will edit the text of the revision with these points in mind.

- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.

As suggested by reviewer, testing additional forms of B cell activation can help explore the generality (or lack thereof) of findings. We plan to test anti-IgM stimulation together with anti-CD40 + BAFF as well as anti-IgM + TLR7/8, and add the data to a revised and final manuscript.

With regards to Fig. 2G (and 2H), in the revised manuscript we will refine the presentation (add a demonstration of the gating, and explicate histogram normalization of FlowJo).

It is an interesting issue in bioscience, but in our presentation 'representative data' really are pretty representative, so a senior author is reminded of a comment Tak Mak made about a reduction (of proliferation, if memory serves) to 0.7 x control. [His point in a comment to referees at a symposium related that to a salary reduction by 30% :) A mathematical alternative is to point out that across four rounds of division for WT cells, a reduction to 0.7x efficiency at each cycle means about 1/4 as many progeny.] 

We will try to edit the revision (Methods, Legends, Results, Discussion] to address better the points of the last two sentences of the comment, and improve the details that could assist in replication or comparisons (e.g., if someone develops a PexRAP inhibitor as potential therapeutic).

For the present, please note that the cell numbers at the end of the cultures are currently shown in Fig 2, panel I. Analogous culture results are shown in Fig 8, panels I, J, albeit with harvesting at day 5 instead of day 4. So, a difference of ≥ 3x needs to be explained. As noted above, a division efficiency reduced to 0.7x normal might account for such a decrease, but in practice the data of Fig. 2I show that the number of PexRAP-deficient B cells at day 4 is similar to the number plated before activation, and yet there has been a reasonable amount of divisions. So cell numbers in the culture of  mutant B cells are constant because cycling is active but decreased and insufficient to allow increased numbers ("proliferation" in the true sense) as programmed death is increased. In line with this evidence, Fig 8G-H document higher death rates [i.e., frequencies of cleaved caspase3⁺ cell and Annexin V⁺ cells] of PexRAP-deficient B cells compared to controls. Thus, the in vitro data lead to the conclusion that both decreased division rates and increased death operate after this form of stimulation.

An inference is that this is the case in vivo as well - note that recoveries differed by ~3x (Fig. 2D), and the decrease in divisions (presentation of which will be improved) was meaningful but of lesser magnitude (Fig. 2E, F).  

Reviewer #2 (Public review):

Summary:

In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.

Strengths:

Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.

We appreciate this positive response and agree with the overview and summary of the paper's approaches and strengths.

Weaknesses:

More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.

Issues about contributions of cell cycling and divisions on the one hand, and susceptibility to death on the other, were discussed above, amplifying on the current manuscript text. The aggregate data support a model in which both processes are impacted for mature B cells in general, and mechanistically the evidence and work focus on the increased ROS and modes of death. Although the data in Fig. 7 do provide evidence that GC B cells themselves are affected, we agree that resource limitations had militated against developing further evidence about cycling specifically for GC B cells. We will hope to be able to obtain sufficient data from some specific analysis of proliferation in vivo (e.g., Ki67 or BrdU) as well as ROS and death ex vivo when harvesting new samples from mice immunized to analyze GC B cells for CXCR4/CD86, CD38, CD138 as indicated by Reviewer 1.  As suggested by Reviewer 2, we will further discuss the possible mechanism(s) by which proliferation of PexRAP-deficient B cells is impaired. We also will edit the text of a revision where to enhance clarity of data interpretation - at a minimum, to be very clear that caution is warranted in assuming that GC B cells will exhibit the same mechanisms as cultures in vitro-stimulated B cells.

The case studies we will be focusing on.

During the event of Jim Crow Laws in 1877 to 1950s, the problems have occurred that crime was functional to be the high associations for a lack of access to quality within increased incarceration levels.

I rely on its exhibition in the era of colorblindness, it was justified for discrimination as in result for insolence must be punished. We shouldn't harm each other.

Cotton's fate of his grandfather's death at the hands of Klan was a tragedy, but he believes his father was trying to prevent form voting by poll taxes and literal expectations.

The Europeans claimed this land was "new". That was not the case, Native Americans had been there for years. It was nothing "new" to them.

The Columbian Exchange was the transfer of goods between the East and the West. This later shape the whole world

Nota para mi: La próxima vez comenzar desde aquí

quitar "como". Opcional: quitar paréntesis.

https://www.facebook.com/groups/721704878218903/posts/2896289730760396/

• Ivan Campos (SoCal)
• Tyler Alan Macek (Bay Area)
• Scott Cuzzo (Ventura)
• Andres Mejia (Fullerton, Orange)

Importante

Paine uses historical and biblical examples to argue that monarchy causes conflict, contrasting peaceful early societies with violent monarchies.

Paine shows the revolution has global significance and urges America to protect liberty, emphasizing the importance of action.

How does Paine's call to action compare to petitions, letters, or other revolutionary writings we've read in the course?

Why does Paine separate society and government? How does this connect to Enlightenment ideas about natural rights or the social contract?

Paine believes America's fight for independence is part of a larger global struggle for freedom, not just a local issue. This helps convince readers that their actions matter worldwide.

Paine argues that when leaders abuse power, people have the right to question and reject them. This shows why colonists could challenge British rules.

Even if people resist new ideas at first, over time most will agree. Paine predicts that support for independence will grow naturally.

He means people working together (society) is good, but government is something we only need because people are not perfect.

"Necessary evil" means something unpleasant but required. Paine argues that even good governments are flawed because they restricted natural freedom.

for those with high autism traits, this "mental rehearsal" system doesn't work as effectively, so their brain doesn't have the same response when just watching.

The author uses the term "flavor" to refer to the core, often unstated, assumptions or a general approach that characterizes traditional cognitive science. This "flavor" includes key implicit commitments that define the framework within which research is conducted.

Pertence ao Município, aos Estados e ao Distrito Federal a titularidade das receitas arrecadadas a título de imposto de renda retido na fonte incidente sobre valores pagos por eles, suas autarquias e fundações a pessoas físicas ou jurídicas contratadas para a prestação de bens ou serviços, conforme disposto nos arts. 158, I, e 157, I, da Constituição Federal. Nesse sentido:

• RE 1293453 - Tema 1.130
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 11/10/2021
• Publicação: 22/10/2021

RECURSO EXTRAORDINÁRIO. REPERCUSSÃO GERAL. INCIDENTE DE RESOLUÇÃO DE DEMANDAS REPETITIVAS (IRDR). DIREITO TRIBUTÁRIO. DIREITO FINANCEIRO. REPARTIÇÃO DE RECEITAS ENTRE OS ENTES DA FEDERAÇÃO. TITULARIDADE DO IMPOSTO DE RENDA INCIDENTE NA FONTE SOBRE RENDIMENTOS PAGOS, A QUALQUER TÍTULO, PELOS MUNICÍPIOS, A PESSOAS FÍSICAS OU JURÍDICAS CONTRATADAS PARA PRESTAÇÃO DE BENS OU SERVIÇOS. ART. 158, INCISO I, DA CONSTITUIÇÃO FEDERAL. RECURSO EXTRAORDINÁRIO DESPROVIDO. TESE FIXADA.

• 1. A Constituição Federal de 1988 rompeu com o paradigma anterior - no qual verificávamos a tendência de concentração do poder econômico no ente central (União)-, implementando a descentralização de competências e receitas aos entes subnacionais, a fim de garantir-lhes a autonomia necessária para cumprir suas atribuições.

• 2. A análise dos dispositivos constitucionais que versam sobre a repartição de receitas entre os Entes Federados, considerando o contexto histórico em que elaborados, deve ter em vista a tendência de descentralização dos recursos e os valores do federalismo de cooperação, com vistas ao fortalecimento e autonomia dos entes subnacionais.

• 3. A Constituição Federal, ao dispor no art. 158, I, que pertencem aos Municípios “ o produto da arrecadação do imposto da União sobre renda e proventos de qualquer natureza, incidente na fonte, sobre rendimentos pagos, a qualquer título, por eles, suas autarquias e pelas fundações que instituírem e mantiverem.”, optou por não restringir expressamente o termo ‘rendimentos pagos’, por sua vez, a expressão ‘a qualquer título’ demonstra nitidamente a intenção de ampliar as hipóteses de abrangência do referido termo. Desse modo, o conceito de rendimentos constante do referido dispositivo constitucional não deve ser interpretado de forma restritiva.

• 4. A previsão constitucional de repartição das receitas tributárias não altera a distribuição de competências, pois não influi na privatividade do ente federativo em instituir e cobrar seus próprios impostos, influindo, tão somente, na distribuição da receita arrecadada, inexistindo, na presente hipótese, qualquer ofensa ao art. 153, III, da Constituição Federal.

• 5. O direito subjetivo do ente federativo beneficiado com a participação no produto da arrecadação do Imposto de Renda Retido na Fonte - IRRF, nos termos dos arts. 157, I, e 158, I, da Constituição Federal, somente existirá a partir do momento em que o ente federativo competente criar o tributo e ocorrer seu fato imponível. No entanto, uma vez devidamente instituído o tributo, não pode a União - que possui a competência legislativa - inibir ou restringir o acesso dos entes constitucionalmente agraciados com a repartição de receitas aos valores que lhes correspondem.

• 6. O acórdão recorrido, ao fixar a tese no sentido de que “O artigo 158, I, da Constituição Federal de 1988 define a titularidade municipal das receitas arrecadadas a título de imposto de renda retido na fonte, incidente sobre valores pagos pelos Municípios, a pessoas físicas ou jurídicas contratadas para a prestação de bens ou serviços”, atentou-se à literalidade e à finalidade (descentralização de receitas) do disposto no art. 158, I, da Lei Maior.

• 7. Ainda que em dado momento alguns entes federados, incluindo a União, tenham adotado entendimento restritivo relativamente ao disposto no art. 158, I, da Constituição Federal, tal entendimento vai de encontro à literalidade do referido dispositivo constitucional, devendo ser extirpado do ordenamento jurídico pátrio.

• 8. A delimitação imposta pelo art. 64 da Lei 9.430/1996 - que permite a retenção do imposto de renda somente pela Administração federal - é claramente inconstitucional, na medida em que cria uma verdadeira discriminação injustificada entre os entes federativos, com nítida vantagem para a União Federal e exclusão dos entes subnacionais.

• 9. Recurso Extraordinário a que se nega provimento. Fixação da seguinte tese para o TEMA 1130: “Pertence ao Município, aos Estados e ao Distrito Federal a titularidade das receitas arrecadadas a título de imposto de renda retido na fonte incidente sobre valores pagos por eles, suas autarquias e fundações a pessoas físicas ou jurídicas contratadas para a prestação de bens ou serviços, conforme disposto nos arts. 158, I, e 157, I, da Constituição Federal.”

Tema 1130 - Titularidade das receitas arrecadadas a título de imposto de renda retido na fonte incidente sobre valores pagos pelos Municípios, suas autarquias e fundações a pessoas físicas ou jurídicas contratadas para a prestação de bens ou serviços.

Tese - Pertence ao Município, aos Estados e ao Distrito Federal a titularidade das receitas arrecadadas a título de imposto de renda retido na fonte incidente sobre valores pagos por eles, suas autarquias e fundações a pessoas físicas ou jurídicas contratadas para a prestação de bens ou serviços, conforme disposto nos arts. 158, I, e 157, I, da Constituição Federal.

Outras ocorrências Decisão (1)

• @hypmod

mensen leren dmv observatie en imitatie

focus op observeerbaar gedrag, objectief.

de persoon analyseert zijn eigen bewustzijn en beschrijft deze

De theorie veronderstelt dat alle aspecten van de mentale toestand van een mens uitsluitend gevormd worden door de functie die zij hebben in de maatschappij

stroming die ervan uit gaat dat menselijk handelen en denken alleen te begrijpen zijn in relatie tot het systeem waarin ze plaatsvinden

eLife Assessment

This paper presents a computational method to infer from data a key feature of affinity maturation: the relationship between the affinity of B-cell receptors and their fitness. The approach, which is based on a simple population dynamics model but inferred using AI-powered Simulation-Based Inference, is novel and valuable. It exploits recently published data on replay experiments of affinity maturation. While the method is well-argued and the validation solid, the potential impact of the study is hindered by its complex presentation, which makes it hard to assess its claims reliably.

Reviewer #1 (Public review):

Summary:

This paper aims to characterize the relationship between affinity and fitness in the process of affinity maturation. To this end, the authors develop a model of germinal center reaction and a tailored statistical approach, building on recent advances in simulation-based inference. The potential impact of this work is hindered by the poor organization of the manuscript. In crucial sections, the writing style and notations are unclear and difficult to follow.

Strengths:

The model provides a framework for linking affinity measurements and sequence evolution and does so while accounting for the stochasticity inherent to the germinal center reaction. The model's sophistication comes at the cost of numerous parameters and leads to intractable likelihood, which are the primary challenges addressed by the authors. The approach to inference is innovative and relies on training a neural network on extensive simulations of trajectories from the model.

Weaknesses:

The text is challenging to follow. The descriptions of the model and the inference procedure are fragmented and repetitive. In the introduction and the methods section, the same information is often provided multiple times, at different levels of detail. This organization sometimes requires the reader to move back and forth between subsections (there are multiple non-specific references to "above" and "below" in the text).

The choice of some parameter values in simulations appears arbitrary and would benefit from more extensive justification. It remains unclear how the "significant uncertainty" associated with these parameters affects the results of inference. In addition, the performance of the inference scheme on simulated data is difficult to evaluate, as the reported distributions of loss function values are not very informative.

Finally, the discussion of the similarities and differences with an alternative approach to this inference problem, presented in Dewitt et al. (2025), is incomplete.

Reviewer #2 (Public review):

Summary:

This paper presents a new approach for explicitly transforming B-cell receptor affinity into evolutionary fitness in the germinal center. It demonstrates the feasibility of using likelihood-free inference to study this problem and demonstrates how effective birth rates appear to vary with affinity in real-world data.

Strengths:

(1) The authors leverage the unique data they have generated for a separate project to provide novel insights into a fundamental question.

(2) The paper is clearly written, with accessible methods and a straightforward discussion of the limits of this model.

(3) Code and data are publicly available and well-documented.

Weaknesses (minor):

(1) Lines 444-446: I think that "affinity ceiling" and "fitness ceiling" should be considered independent concepts. The former, as the authors ably explain, is a physical limitation. This wouldn't necessarily correspond to a fitness ceiling, though, as Figure 7 shows. Conversely, the model developed here would allow for a fitness ceiling even if the physical limit doesn't exist.

(2) Lines 566-569: I would like to see this caveat fleshed out more and perhaps mentioned earlier in the paper. While relative affinity is far more important, it is not at all clear to me that absolute affinity can be totally ignored in modeling GC behavior.

(3) One other limitation that is worth mentioning, though beyond the scope of the current work to fully address: the evolution of the repertoire is also strongly shaped by competition from circulating antibodies. (Eg: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600904/, http://www.sciencedirect.com/science/article/pii/S1931312820303978). This is irrelevant for the replay experiment modeled here, but still an important factor in general repertoires.

eLife Assessment

This valuable study proposes a theoretical model of clathrin coat formation based on membrane elasticity that seeks to determine whether this process occurs by increasing the area of a protein-coated patch with constant curvature, or by increasing the curvature of a protein-coated patch that forms in an initially flat conformation (so called constant curvature or constant area models). Identifying energetically favorable pathways and comparing the obtained shapes with experiments provides solid support to the constant-area pathway. This work will be of interest for biologists and biophysicists interested in membrane remodelling and endocytosis. It provides an innovative approach to tackle the question of constant curvature vs. constant area coat protein formation, although some of the model's assumption are only partially supported by experimental evidence.

Reviewer #1 (Public review):

Summary:

The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.

Strengths:

The models that the authors choose are fairly well-described in the field and the manuscript is well-written.

Weaknesses:

One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.

While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.

Comments on revisions:

I appreciate the authors edits, but I found that the major concerns I had still hold. Therefore, I did not alter my review.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors employ theoretical analysis of an elastic membrane model to explore membrane vesiculation pathways in clathrin-mediated endocytosis. A complete understanding of clathrin-mediated endocytosis requires detailed insight into the process of membrane remodeling, as the underlying mechanisms of membrane shape transformation remain controversial, particularly regarding membrane curvature generation. The authors compare constant area and constant membrane curvature as key scenarios by which clathrins induce membrane wrapping around the cargo to accomplish endocytosis. First, they characterize the geometrical aspects of the two scenarios and highlight their differences by imposing coating area and membrane spontaneous curvature. They then examine the energetics of the process to understand the driving mechanisms behind membrane shape transformations in each model. In the latter part, they introduce two energy terms: clathrin assembly or binding energy, and curvature generation energy, with two distinct approaches for the latter. Finally, they identify the energetically favorable pathway in the combined scenario and compare their results with experiments, showing that the constant-area pathway better fits the experimental data.

Strengths:

The manuscript is well-written, well-organized, and presents the details of the theoretical analysis with sufficient clarity.<br /> The calculations are valid, and the elastic membrane model is an appropriate choice for addressing the differences between the constant curvature and constant area models.<br /> The authors' approach of distinguishing two distinct free energy terms-clathrin assembly and curvature generation-and then combining them to identify the favorable pathway is both innovative and effective in addressing the problem.<br /> Notably, their identification of the energetically favorable pathways, and how these pathways either lead to full endocytosis or fail to proceed due to insufficient energetic drives, is particularly insightful.

Comments on revisions:

The authors have carefully addressed all my comments, and the revised manuscript is now clear, rigorous, and satisfactory.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1:

Summary

The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.

Strengths

The models that the authors choose are fairly well-described in the field and the manuscript is wellwritten.

Thank you for your positive comments on our work.

Weaknesses

One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.

Thank you for your insightful questions. We fully agree that distinguishing between the two models should ultimately be guided by experimental tests. This is precisely the motivation for including Fig. 5 in our manuscript, where we compare our theoretical predictions with experimental data. In the middle panel of Fig. 5, we observe that the predicted tip radius as a function of 𝜓_𝑚𝑎𝑥 from the constant curvature model (magenta curve) deviates significantly from both the experimental data points and the rolling median, highlighting the inconsistency of this model with the data.

Regarding our treatment of clathrin assembly and membrane adhesion as mechanical equilibrium processes, our reasoning is based on a timescale separation argument. Clathrin assembly typically occurs over approximately 1 minute. In contrast, the characteristic relaxation time for a lipid membrane to reach mechanical equilibrium is given by , where 𝜇∼5 × 10^-9 𝑁𝑠𝑚^-1 is the membrane viscosity, 𝑅₀ =50𝑛𝑚 is the vesicle size, 𝜅=20 𝑘_𝐵𝑇 is the bending rigidity. This yields a relaxation time of 𝜏≈1.5 × 10⁻⁴𝑠, which is several orders of magnitude shorter than the timescale of clathrin assembly. Therefore, it is reasonable to treat the membrane shape as being in mechanical equilibrium throughout the assembly process.

We believe the value of our model lies in the following key novelties:

(1) Model novelty: We introduce an energy term associated with curvature generation, a contribution that is typically neglected in previous models.

(2) Methodological novelty: We perform a quantitative comparison between theoretical predictions and experimental data, whereas most earlier studies rely on qualitative comparisons.

(3) Results novelty: Our quantitative analysis enables us to unambiguously exclude the constant curvature hypothesis based on time-independent electron microscopy data.

In the revised manuscript (line 141), we have added a statement about why we treat the clathrin assembly as in mechanical equilibrium.

While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.

Author response: We thank the reviewer for raising this important point. We agree that there is uncertainty in the experimental data. Our decision to symmetrize the data is based on the following considerations:

(1) The experimental data provide a one-dimensional membrane profile corresponding to a cross-sectional view. To reconstruct the full two-dimensional membrane surface, we must assume rotational symmetry.

(2)In addition to symmetrization, we also average membrane profiles within a certain range of 𝜓_𝑚𝑎𝑥 values (see Fig. 5d). This averaging helps reduce the uncertainty (due to biological and experimental variability) inherent to individual measurements.

(3)To further address the noise in the experimental data, we compare our theoretical predictions not only with individual data points but also with a rolling median, which provides a smoothed representation of the experimental trends.

These steps are taken to ensure a more robust and meaningful comparison between theory and experiments.

In the revised manuscript (line 338), we have explained why we have to symmetrize the data:

“To facilitate comparison between the axisymmetric membrane shapes predicted by the model and the non-axisymmetric profiles obtained from electron microscopy, we apply a symmetrization procedure to the experimental data, which consist of one-dimensional membrane profiles extracted from cross-sectional views, as detailed in Appendix 3 (see also Appendix 3--Fig. 1).”

Reviewer #2:

Summary

In this manuscript, the authors employ theoretical analysis of an elastic membrane model to explore membrane vesiculation pathways in clathrin-mediated endocytosis. A complete understanding of clathrin-mediated endocytosis requires detailed insight into the process of membrane remodeling, as the underlying mechanisms of membrane shape transformation remain controversial, particularly regarding membrane curvature generation. The authors compare constant area and constant membrane curvature as key scenarios by which clathrins induce membrane wrapping around the cargo to accomplish endocytosis. First, they characterize the geometrical aspects of the two scenarios and highlight their differences by imposing coating area and membrane spontaneous curvature. They then examine the energetics of the process to understand the driving mechanisms behind membrane shape transformations in each model. In the latter part, they introduce two energy terms: clathrin assembly or binding energy, and curvature generation energy, with two distinct approaches for the latter. Finally, they identify the energetically favorable pathway in the combined scenario and compare their results with experiments, showing that the constant-area pathway better fits the experimental data.

Thank you for your clear and comprehensive summary of our work.

Strengths

The manuscript is well-written, well-organized, and presents the details of the theoretical analysis with sufficient clarity. The calculations are valid, and the elastic membrane model is an appropriate choice for addressing the differences between the constant curvature and constant area models.

The authors' approach of distinguishing two distinct free energy terms-clathrin assembly and curvature generation-and then combining them to identify the favorable pathway is both innovative and effective in addressing the problem.

Notably, their identification of the energetically favorable pathways, and how these pathways either lead to full endocytosis or fail to proceed due to insufficient energetic drives, is particularly insightful.

Thank you for your positive remarks regarding the innovative aspects of our work.

Weaknesses and Recommendations

Weakness: Membrane remodeling in cellular processes is typically studied in either a constant area or constant tension ensemble. While total membrane area is preserved in the constant area ensemble, membrane area varies in the constant tension ensemble. In this manuscript, the authors use the constant tension ensemble with a fixed membrane tension, σe. However, they also use a constant area scenario, where 'area' refers to the surface area of the clathrin-coated membrane segment. This distinction between the constant membrane area ensemble and the constant area of the coated membrane segment may cause confusion.

Recommendation: I suggest the authors clarify this by clearly distinguishing between the two concepts by discussing the constant tension ensemble employed in their theoretical analysis.

Thank you for raising this question.

In the revised manuscript (line 136), we have added a sentence, emphasizing the implication of the term “constant area model”:

“We emphasize that the constant area model refers to the assumption that the clathrin-coated area 𝑎₀ remains fixed. Meanwhile, the membrane tension 𝜎_𝑒 at the base is held constant, allowing the total membrane area 𝐴𝐴 to vary in response to deformations induced by the clathrin coat.”

Weakness: As mentioned earlier, the theoretical analysis is performed in the constant membrane tension ensemble at a fixed membrane tension. The total free energy E_tot of the system consists of membrane bending energy E_b and tensile energy E_t, which depends on membrane tension, σe. Although the authors mention the importance of both E_b and E_t, they do not present their individual contributions to the total energy changes. Comparing these contributions would enable readers to cross-check the results with existing literature, which primarily focuses on the role of membrane bending rigidity and membrane tension.

Recommendation: While a detailed discussion of how membrane tension affects their results may fall outside the scope of this manuscript, I suggest the authors at least discuss the total membrane area variation and the contribution of tensile energy E_t for the singular value of membrane tension used in their analysis.

Thank you for the insightful suggestion. In the revised manuscript (line 916), we have added Appendix 6 and a supplementary figure to compare the bending energy 𝐸_𝑏 and the tension energy 𝐸_𝑡. Our analysis shows that both energy components exhibit an energy barrier between the flat and vesiculated membrane states, with the tension energy contributing more significantly than the bending energy.

In the revised manuscript (line 151), we have also added one paragraph explaining why we set the dimensionless tension . This choice is motivated by our use of the characteristic length as the length scale, and as the energy scale. In this way, the dimensionless tension energy is written as

Where is the dimensionless area.

Weakness: The authors introduce two different models, (1,1) and (1,2), for generating membrane curvature. Model 1 assumes a constant curvature growth, corresponding to linear curvature growth, while Model 2 relates curvature growth to its current value, resembling exponential curvature growth. Although both models make physical sense in general, I am concerned that Model 2 may lead to artificial membrane bending at high curvatures. Normally, for intermediate bending, ψ > 90, the bending process is energetically downhill and thus proceeds rapidly. The bending process is energetically downhill and thus proceeds rapidly. However, Model 2's assumption would accelerate curvature growth even further. This is reflected in the endocytic pathways represented by the green curves in the two rightmost panels of Fig. 4a, where the energy steeply increases at large ψ. I believe a more realistic version of Model 2 would require a saturation mechanism to limit curvature growth at high curvatures.

Recommendation 1: I suggest the authors discuss this point and highlight the pros and cons of Model 2. Specifically, addressing the potential issue of artificial membrane bending at high curvatures and considering the need for a saturation mechanism to limit excessive curvature growth. A discussion on how Model 2 compares to Model 1 in terms of physical relevance, especially in the context of high curvature scenarios, would provide valuable insights for the reader.

Thank you for raising the question of excessive curvature growth in our models and the constructive suggestion of introducing a saturation mechanism. In the revised manuscript (line 405), following your recommendation, we have added a subsection “Saturation effect at high membrane curvatures” in the discussion to clarify the excessive curvature issue and a possible way to introduce a saturation mechanism:

“Note that our model involves two distinct concepts of curvature growth. The first is the growth of imposed curvature — referred to here as intrinsic curvature and denoted by the parameter 𝑐₀ — which is driven by the reorganization of bonds between clathrin molecules within the coat. The second is the growth of the actual membrane curvature, reflected by the increasing value of 𝜓_𝑚𝑎𝑥.

The latter process is driven by the former.

Models (1,1) and (1,2) incorporate energy terms (Equation 6) that promote the increase of intrinsic curvature 𝑐₀, which in turn drives the membrane to adopt a more curved shape (increasing 𝜓_𝑚𝑎𝑥). In the absence of these energy contributions, the system faces an energy barrier separating a weakly curved membrane state (low 𝜓_𝑚𝑎𝑥) from a highly curved state (high 𝜓_𝑚𝑎𝑥). This barrier can be observed, for example, in the red curves of Figure 3(a–c) and in Appendix 6—Figure 1. As a result, membrane bending cannot proceed spontaneously and requires additional energy input from clathrin assembly.

The energy terms described in Equation 6 serve to eliminate this energy barrier by lowering the energy difference between the uphill and downhill regions of the energy landscape. However, these same terms also steepen the downhill slope, which may lead to overly aggressive curvature growth.

To mitigate this effect, one could introduce a saturation-like energy term of the form:

where 𝑐_𝑠 represents a saturation curvature. Importantly, adding such a term would not alter the conclusions of our study, since the energy landscape already favors high membrane curvature (i.e., it is downward sloping) even without the additional energy terms. “

Recommendation 2: Referring to the previous point, the green curves in the two rightmost panels of Fig. 4a seem to reflect a comparison between slow and fast bending regimes. The initial slow vesiculation (with small curvature growth) in the left half of the green curves is followed by much more rapid curvature growth beyond a certain threshold. A similar behavior is observed in Model 1, as shown by the green curves in the two rightmost panels of Fig. 4b. I believe this transition between slow and fast bending warrants a brief discussion in the manuscript, as it could provide further insight into the dynamic nature of vesiculation.

Thank you for your constructive suggestion regarding the transition between slow and fast membrane bending. As you pointed out, in both Fig. 4a (model (1,2)) and Fig. 4b (model (1,1)), the green curves tend to extend vertically at the late stage. This suggests a significant increase in 𝑐₀ on the free energy landscape. However, we remain cautious about directly interpreting this vertical trend as indicative of fast endocytic dynamics, since our model is purely energetic and does not explicitly incorporate kinetic details. Meanwhile, we agree with your observation that the steep decrease in free energy along the green curve could correspond to an acceleration in dynamics. To address this point, we have added a paragraph in the revised manuscript (in Subsection “Cooperativity in the curvature generation process”) discussing this potential transition and its consistency with experimental observations (line 395):

“Furthermore, although our model is purely energetic and does not explicitly incorporate dynamics, we observe in Figure 3(a) that along the green curve—representing the trajectory predicted by model (1,2)—the total free energy (𝐸_𝑡𝑜𝑡) exhibits a much sharper decrease at the late stage (near the vesiculation line) compared to the early stage (near the origin). This suggests a transition from slow to fast dynamics during endocytosis. Such a transition is consistent with experimental observations, where significantly fewer number of images with large 𝜓_𝑚𝑎𝑥 are captured compared to those with small 𝜓_𝑚𝑎𝑥 (Mund et al., 2023).”

The geometrical properties of both the constant-area and constant-curvature scenarios, as well depicted in Fig. 1, are somewhat straightforward. I wonder what additional value is presented in Fig. 2. Specifically, the authors solve differential shape equations to show how Rt and Rcoat vary with the angle ψ, but this behavior seems predictable from the simple schematics in Fig. 1. Using a more complex model for an intuitively understandable process may introduce counter-intuitive results and unnecessary complications, as seen with the constant-curvature model where Rt varies (the tip radius is not constant, as noted in the text) despite being assumed constant. One could easily assume a constant-curvature model and plot Rt versus ψ. I wonder What is the added value of solving shape equations to measure geometrical properties, compared to a simpler schematic approach (without solving shape equations) similar to what they do in App. 5 for the ratio of the Rt at ψ=30 and 150.

Thank you for raising this important question. While simple and intuitive theoretical models are indeed convenient to use, their validity must be carefully assessed. The approximate model becomes inaccurate when the clathrin shell significantly deviates from its intrinsic shape, namely a spherical cap characterized by intrinsic curvature 𝑐₀. As shown in the insets of Fig. 2b and 2c (red line and black points), our comparison between the simplified model and the full model demonstrates that the simple model provides a good approximation under the constant-area constraint. However, it performs poorly under the constant-curvature constraint, and the deviation between the full model and the simplified model becomes more pronounced as 𝑐₀ increases.

In the revised manuscript, we have added a sentence emphasizing the discrepancy between the exact calculation with the idealized picture for the constant curvature model (line 181):

“For the constant-curvature model, the ratio remains close to 1 only at small values of 𝑐₀, as expected from the schematic representation of the model in Figure 1. However, as 𝑐₀ increases, the deviation from this idealized picture becomes increasingly pronounced.”

Recommendation: The clathrin-mediated endocytosis aims at wrapping cellular cargos such as viruses which are typically spherical objects which perfectly match the constant-curvature scenario. In this context, wrapping nanoparticles by vesicles resembles constant-curvature membrane bending in endocytosis. In particular analogous shape transitions and energy barriers have been reported (similar to Fig.3 of the manuscript) using similar theoretical frameworks by varying membrane particle binding energy acting against membrane bending:

DOI: 10.1021/la063522m

DOI: 10.1039/C5SM01793A

I think a short comparison to particle wrapping by vesicles is warranted.

Thank you for your constructive suggestion to compare our model with particle wrapping. In the revised manuscript (line 475), we have added a subsection “Comparison with particle wrapping” in the discussion:

“The purpose of the clathrin-mediated endocytosis studied in our work is the recycling of membrane and membrane-protein, and the cellular uptake of small molecules from the environment — molecules that are sufficiently small to bind to the membrane or be encapsulated within a vesicle. In contrast, the uptake of larger particles typically involves membrane wrapping driven by adhesion between the membrane and the particle, a process that has also been studied previously (Góźdź, 2007; Bahrami et al., 2016). In our model, membrane bending is driven by clathrin assembly, which induces curvature. In particle wrapping, by comparison, the driving force is the adhesion between the membrane and a rigid particle. In the absence of adhesion, wrapping increases both bending and tension energies, creating an energy barrier that separates the flat membrane state from the fully wrapped state. This barrier can hinder complete wrapping, resulting in partial or no engulfment of the particle. Only when the adhesion energy is sufficiently strong can the process proceed to full wrapping. In this context, adhesion plays a role analogous to curvature generation in our model, as both serve to overcome the energy barrier. If the particle is spherical, it imposes a constant-curvature pathway during wrapping. However, the role of clathrin molecules in this process remains unclear and will be the subject of future investigation.”

Minor points:

Line 20, abstract, "....a continuum spectrum ..." reads better.

Line 46 "...clathrin results in the formation of pentagons ...." seems Ito be grammatically correct.

Line 106, proper citation of the relevant literature is warranted here.

Line 111, the authors compare features (plural) between experiments and calculations. I would write "....compare geometric features calculated by theory with those ....".

Line 124, "Here, we choose a ..." (with comma after Here).

Line 134, "The membrane tension \sigma_e and bending rigidity \kappa define a ...."

Line 295, "....tip radius, and invagination ...." (with comma before and).

Line 337, "abortive tips, and ..." (with comma before and).

We thank you for your thorough review of our manuscript and have corrected all the issues raised.

In such terms I expected, summary and identifications can respond to our text, and some case, readers can be able to review a different topic on their own statement.

Critique was pronounced that is official, but it is unclear that you will be said to respond to your writer without listening deeply.

These views can be important, but difficult, however, to those who responds the language, the concepts of the text adheres their consideration.

With the lack of discussion, readers cannot understand to build arguments that can never read through conversation.

In one big mistake to read the passage in the wrong way, it will be denied. The author is questioning about whether most Americans obtain the ticket rather than ticket to middle-class security in a word.

According to my opinion, "although many believe" is too clear for any observations for road-mapping phrase.

I imagine the multisided statement is to persuade the agreement and make an offer for ongoing request that they're up for. It can be a serious claim what I have known.

It is unclear that why did the provocation of this argument responds to any students, but the results are often impacted in undisclosed measures.

At the same time, a challenge can be resourceful and important to justify the result to our stratagem.

As I assure the discussion, this thesis can be often essential for a summary, according to a author's central thesis task.

we need to go beyond ethical protocols and into active decolonization

colonial past implications for reciprocity practice

the political problem. How to go beyond academic reciprocity and into social reciprocity.

Similar to the concept of agential realism!

key term here: "ancestors". We need to think of reciprocity in a way that account for the years of exploitation of our ancestors.

This should be in the RESULTS!

Should it be larva??

Didn't identify HOW they evoked this behavior though

Seems like a very bold statement

Would hesitate to call this change simple

Not correct

Cite more, This bibliography is not substantial. once again look at the novet catalogue

Perhaps comment on what this is, analyze it maybe

a lot to talk about, maybe do some more research on "Brygos Painter". This is a very short article, try finding more information on the Novet catalogue

2_mypy.mdを3_mypy.mdにリネームすれば5.2.になります。このPRでは2_mypy.mdは触らないほうがいいと思うので、このままにしておきます。

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OpenAI Dev Day 2025: AgentKit & Platform Strategy

Overview & Platform Vision

• OpenAI positions developers as the distribution layer for AGI benefits: > "our mission at OpenAI is to, one, build AGI...and then...just as important is to bring the benefits of that to the entire world...we really need to rely on developers, other third parties to be able to do this"
• Developer ecosystem growth: 4 million developers (up from ~3 million last year)
• ChatGPT now 5th or 6th largest website globally with 800 million weekly active users

• "Today we're going to open up ChatGPT for developers to build real apps inside of ChatGPT...with the Apps SDK, your apps can reach hundreds of millions of ChatGPT users" — Sam Altman

Major Model Releases

API Parity with Consumer Products: - GPT-5 Pro - flagship model now available via API - Sora 2 & Sora 2 Pro - video generation models released - Distilled models: - gpt-realtime-mini (70% cheaper) - gpt-audio-mini - gpt-image-1-mini (80% cheaper)

Apps SDK & MCP Integration

• Built on Model Context Protocol (MCP), first major platform to adopt it

• "OpenAI adopted [MCP] so quickly, much less to now be the first to turn it into the basis of a full app store platform"

• Technical innovations:
• React component bundling for iframe targets with custom UI components
• Live data flow (demonstrated with Coursera app allowing queries during video watching)
• OpenAI joined MCP steering committee in March 2025, with Nick Cooper as representative

• "they really treat it as an open protocol...they are not viewing it as this thing that is specific to Anthropic"

AgentKit Platform Components

Agent Builder

• Visual workflow builder with drag-and-drop interface

• "launched agent kit today, full set of solutions to build, deploy and optimize agents"

• Supports both deterministic and LLM-driven workflows
• Uses Common Expression Language (CEL) for conditional logic
• Features: user approval nodes, transform/set state capabilities, templating system
• Pre-built templates: customer support, document discovery, data enrichment, planning helper, structured data Q&A, document comparison, internal knowledge assistant

Agent SDK

• "allowing you to use [traces] in the evals product and be able to grade it...over the entirety of what it's supposed to be doing"

• Supports MCP protocol integration
• Enables code export from Agent Builder for standalone deployment
• Built-in tracing capabilities for debugging and evaluation

ChatKit

• Consumer-grade embeddable chat interface

• "ChatKit itself is like an embeddable iframe...if you are using ChatKit and we come up with new...a new model that reasons in a different way...you don't actually need to rebuild"

• Designed by team that built Stripe Checkout
• Provides "full stack" with widgets and custom UI components
• Already powers help.openai.com customer support

Connector Registry

• First-party "sync connectors" that store state for re-ranking and optimization
• Third-party MCP server support

• "we end up storing quite a bit of state...we can actually end up doing a lot more creative stuff...when you're chatting with ChatGPT"

• Tradeoffs between first-party depth vs third-party breadth discussed

Evaluation Tools

• Agent-specific eval capabilities for multi-step workflows

• "how do you even evaluate a 20 minute task correctly? And it's like, it's a really hard problem"

• Multi-model support including third-party models via OpenRouter integration
• Automated prompt optimization with LM-as-judge rubrics
• Future plans for component-level evaluation of complex traces

Developer Experience Insights

Prompt Engineering Evolution

• "two years ago people were like, oh, at some point...prompting is going to be dead...And if anything, it is like become more and more entrenched"

• Research advancing with GEPA (Databricks) and other optimization techniques

• "it is like pretty difficult for us to manage all of these different [fine-tuning] snapshots...if there is a way to...do this like zero gradient like optimization via prompts...I'm all for it"

Internal Codex Usage

• Agent Builder built in under 2 months using Codex

• "on their way to work, they're like kicking off like five Codex tasks because the bus takes 30 minutes...and it kind of helps you orient yourself for the day"

• High-quality PR reviews from Codex widely adopted internally
• Pattern shift: > "push yourself to like trust the model to do more and more...full YOLO mode, like trust it to like write the whole feature"

Infrastructure & Reliability

Service Health Dashboard

• New org-scoped SLO tracking for API integrations
• Monitors token velocity (TPM), throughput, response codes in real-time

• "We haven't had one [major outage] that bad since...We think we've got reliability in a spot where we're comfortable kind of putting this out there"

• Target: moving from 4 nines toward 5 nines availability (exponentially more work per nine)
• Serving >6 billion tokens per minute (stat already outdated at time of interview)

Strategic Partnerships

• Apple Siri integration: ChatGPT account status determines model routing (free vs Plus/Pro)
• Kakao (Korea's largest messenger app): Sign-in with ChatGPT integration
• Jony Ive and Stargate announcements happening offstage

Key Personalities

• Sherwin Wu - Head of Engineering, OpenAI Platform
• Christina Huang - Platform Experience, OpenAI
• John Schulman - Now at xAI, launched Tinker API (low-level fine-tuning library he championed at both OpenAI and Anthropic)
• Michelle Pokrass - Former API team (2024), championed "API = AGI" philosophy
• Greg Brockman - Mentioned sustainable businesses built on Custom GPTs
• Sam Altman - Delivered keynote, announced Apps SDK

References & Tools

• OpenAI Platform Docs
• OpenAI Cookbook - Sora 2 Prompting Guide
• chatkit.studio - Playground and demos
• widget.studio - Widget builder
• chatkit.world - Interactive demo site
• Ben Thompson's endorsement of Christina's demo
• Model Context Protocol (MCP) consortium

Future Directions

• Multimodal evals expansion
• Voice modality for Agent Builder
• Human-in-the-loop workflows over weeks, not just binary approvals
• Bring-your-own-key (BYOK) for public agent deployments
• Protocol standardization (responses API, agent workflows)
• Enhanced widget ecosystem potentially user-contributed

• rechter grijpt alleen in als het besluit duidelijk onredelijk, onrechtmatig of in strijd met de ABBB is

• gaan over de inhoud van het besluit zelf, dus wat er besloten wordt
• ze zorgen ervoor dat het besluit rechtvaardig, proportioneeel en in overeenstemming met de wet is

• gaan over de manier waarop een besluit tot stand komt
• ze zorgen ervoor dat de overheid zorgvuldig, eerlijk en transparant handelt bij het nemen van besluiten

• gaan over de inhoud van het besluit zelf - dus wat er beslist is
• een fout in de inhoud van het besluit

• als een burger redelijkerwijs mag vertrouwen op een uitspraak, toezegging of gedraging van de overheid, dan moet de overheid dat vertrouwen in principe nakomen
• overheid moet betrouwbaar en voorspelbaar zijn in wat ze zegt en doet

• overheid moet gelijke gevallen gelijk behandelen, en ongelijke gevallen ongelijk
• burgers die in dezelfde situatie verkeren moeten hetzelfde behandeld worden door de overheid

• goed onderbouwd en overtuigend

houdt in dat - de argumenten inhoudelijk sterk genoeg zijn - ze logisch voortvloeien uit de feiten - en ze de beslissing echt kunnen dragen